1. Field of the Invention
The present invention relates to the activation of certain polysaccharide matrices for use in affinity chromatography procedures, and, more especially, to the sodium metaperiodate [NaIO.sub.4 ] activation of polysaccharide matrices, to certain polyhydrazide derivatives thereof, to the "reductive stabilization" thereof and of such polyhydrazide derivatives, and to the coupling of various biologically active molecules thereto as well as to such polyhydrazide derivatives.
2. Description of the Prior Art
Affinity chromatography has found wide application in the purification of various biologically active molecules, such as small ligands, proteins, nucleotides and nucleosides. For example, nucleotides are cofactors and coenzymes in many biological systems and, therefore, many methods for attaching these compounds to insoluble matrices have been developed and applied. It is also known that certain polysaccharide matrices comprise the most useful solid supports in affinity chromatography. And various alternative methods exist to activate a polysaccharide matrix, e.g., cellulose, starch and cross-linked polysaccharide gels such as agarose, Sephadex and Sepharose, for the covalent attachment of, e.g., small ligands and proteins. Probably the most widely used technique at the present time for the covalent coupling of protein to insoluble matrices, finding considerable application in immunology and enzymology, is the cyanogen bromide method described in Axen et al, Nature, 214, 1302-1304 (1967); see also Cuatrecasas et al, Proc. Natl. Acad. Sci. U.S., 61, 636-643 (1968). Briefly, cyanogen halides react rapidly with the hydroxyl groups of carbohydrates to form cyanate esters. The reaction occurs most rapidly at a pH between 10 and 12. The cyanate may react further with hydroxyl groups to form an imidocarbonate intermediate. It has been postulated from studies of low molecular weight model compounds, that the cyclic imidocarbonate is formed as a major product following the reaction of cyanogen halides with polymeric carbohydrates [FIG. 1A]. The highly reactive intermediate can be isolated, and agarose beads which contain this reactive group are commerically available [Pharmacia]. Imidocarbonates react with amines to form N-imidocarbonates [FIG. 1B], isoureas [FIG. 1C], or N-carbamates [FIG. 1D]. However, the actual products formed in the reaction between cyanogen halide-activated agarose and the amino group of alanine, for example, are difficult to study owing to the insolubility of the carbohydrate and the relatively low concentrations of the products. Recent studies on the isoelectric point of products of the reaction between CNBr-activated polysaccharides and a primary amine indicate that an isourea is the major product, Svensson, FEBS Lett., 29, 167 (1973). The technique of Axen et al, supra, characterizes the "titration method" of activation.
Despite the relative simplicity of the titration method of activation of agarose beads, a faster and safer alternative method has been developed. Cuatrecasas et al, Anal. Biochem., in press. In this modification, the reaction is performed in carbonate buffer. The coupling efficiencies obtained are comparable to those observed after activation by the aforementioned titration method.
Generally considering the use of CNBr-activated gels, the amount of ligand coupled to the gel depends on the amount of CNBr added. Typically, this varies between 50 and 300 mg of CNBr per milliliter of packed beads. For example, with 200 mg of CNBr per milliliter of agarose and 0.5% albumin and 0.2 M NaHCO.sub.3 at pH 9.5, approximately 5 mg of protein is bound per milliliter of packed gel. Similarly, if the concentration of low molecular weight ligand, e.g., alanine, is 0.1 M, the amount coupled is about 10 .mu. moles per milliliter of gel. The actual coupling efficiency will depend on the specific ligand used.
The quantity of CNBr and the exact composition of the buffer used in the coupling reaction should be adapted to the specific system under study. These conditions too have been described in detail, Cuatrecasas, J. Biol. Chem., 245, 3059 (1970). A standard condition is the use of 200 mg of CNBr per milliliter of agarose, and of 0.2 M sodium bicarbonate at pH 9.5 as the buffer for the coupling reaction. Smaller quantities of CNBr, lower pH values, and high concentrations of ligand will decrease the probability of multipoint attachments of proteins [especially those of high molecular weight] to the gel, a condition that may lead to decreased or altered biological activity. In coupling macromolecules, e.g., concanavalin A or enzymes, it may be desirable to add ligands, e.g., competitive inhibitors, which bind to the active site of the protein. This may protect and perhaps prevent coupling through essential residues of the active site, and in addition, may stabilize the conformation of the protein so that the coupled protein will be more likely to retain its native structure.
In many cases, the interposition of spacers or "arms" between the matrix and the ligand greatly increases the effectiveness of the adsorbent. A variety of spacer molecules can be attached to agarose, and many chemical reactions exist that can be used to couple ligands and proteins to these derivatized agarose gels, Cuatrecasas, J. Biol. Chem., supra. Diaminodipropylamine [Eastman] has been one of the most useful spacer molecules because it is relatively long and because it exhibits very minimal hydrophobic properties as compared to strictly methylenic diamine compounds such as hexamethylenediamine. Consider also copending application, Ser. No. 475,314 of the present inventors, filed concurrently herewith, and hereby expressly incorporated by reference. Whenever possible, it is advantageous to first attach such spacers to the ligand rather than to the gel since the adsorbents prepared in this way are less likely to exhibit nonspecific or ionic properties that can interfere in subsequent affinity chromatography experiments.
Nevertheless, CNBr is a toxic chemical, and awareness of the hazards associated with CNBr activation reactions is important. CNBr, which sublimes rather rapidly at room temperature, is a powerful lachrimator, and is highly toxic. Allowing the chemical to stand for months at room temperature can result in the formation of explosive compounds. Filtrates collected after activation contain large amounts of cyanide ion and should never be allowed to come to neutral or acidic pH. Disposal of the filtrates should be performed with care and only after the gaseous products are disssipated from the filter flask in a fume hood for several hours. The importance of adequate ventilation during the activation procedures has been emphasized. Another disadvantage of the CNBr method for coupling amino group-containing ligands to agarose lies in the relative instability of the bond(s) formed between the ligand and the matrix. As a result, in most cases, during washing, storage and usage, a small amount of ligand is leaked out. This problem of ligand leakage is of great importance, especially during isolation of small [microgram] quantities of certain hormone receptors. The leakage of a ligand from the adsorbent becomes still more serious because the ligand which is released from the adsorbent in most cases has greater affinity toward the receptor protein(s) than the matrix-bound ligand. Besides the problem of ligand leakage, substitution does not work well with cellulose.
While nearly all of the earlier methods for coupling ligands or proteins to agarose depend on the initial modification of the gel with CNBr, an alternative method exists which is rapid, simple, and safe and which promises to result in chemically stable ligand-agarose bonds. This method depends on the oxidation of cis-vicinal hydroxyl groups of agarose [or cellulose] by sodium metaperiodate [NaIO.sub.4 ] to generate aldehyde functions. Compare Sanderson et al, Immunology, 20, 1061-1065 (1971); see FIG. 2. These aldehydic functions react at pH 4-6 with primary amines to form Schiff bases [aldimines]; these are reduced with sodium borohydride [NaBH.sub.4 ] to form stable secondary amines.